Studies on the unfolding and zinc status of thermolysin and carboxypeptidase A, and the design of a fret-quenched peptide for assaying thermolysin-like proteases
Year: 2020
Type: dissertation
Abstract: The chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) has previously been \nemployed as a probe to determine structural transitions during protein (un)folding in the zincdependent anthrax lethal factor protease (LF). Whether PAR can be employed more widely as a \nzinc accessibility probe (ZAP) to study the unfolding of other zinc proteins remains to be \nestablished. Using a combination of intrinsic tryptophan fluorescence spectroscopy and chelator \nstudies, the unfolding pathways and the influence of the protein fold on the metal status were \ninvestigated for the zinc proteases thermolysin (TL) and carboxypeptidase A (CPA). These studies \nrevealed considerable differences in the unfolding pathways and the resistance to metal \ndissociation for these enzymes. In addition, the observations reported herein demonstrate that \nZAPs might be of value in future investigations to gain further insight into the mechanisms \nunderlying the metal-mediated misfolding of proteins involved in a variety of neurodegenerative \ndiseases. Finally, this study describes a new fluorescence-quenched heptapeptide \n(Dabcyl-FKFLGKE-EDANS), which shows the largest specificity constant documented for any TL \nsubstrate.
Author: Danica L. Goulet
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3131643708